Sigma receptors ligands with anti-apoptotic and/or pro-apoptotic properties, over cellular mechanisms, exhibiting prototypical cytoprotective and also anticancer activity

ABSTRACT

The present invention involves new and original sigma receptors Ligands: (Mono-or dialkylaminoalkyl)-γ-butyro-lactones, their analogues aminotetrahydroturanes, the (1-adamantyl) benzene alkylamines, the N,N Dialkyl α-[(adamantyl-1)ben-zyloxy-2] alkylamines and the 3-cyclopentyl adamantyl-amines or alkylamines or-alkyl phenylamines, their enantiomers or di-astereoisomers, their pharmaceutically acceptable salts and Quinacrine Me-thylene blue, Astemizole and their relative analogues with pro-apoptotic and/or anti-apoptotic properties over cellular biochemical mechanisms, with prototypical anti-cancer, an-timetastatic and antiviral activities associated with antagonism of the neuropatic pain and, at very low doses, with cytoprotectve and cytoregenerative activity against the cytodegenerative diseases.

The present invention concerns the prototypical a receptor ligands(Mono- or Dialky-laminoalkyl)-γ-butyrolactones (AL), their analoguesaminotetrahydrofuranes (AE), with various substitutes: phenyl-,diphenyl-, phenoxymethyl, fluorenyl or adamantyl, the (1-adamantyl)phenyl(s) alkylamines-(AdBAA), the N,N Dialkyl α-[(adamantyl-1)benzyloxy-2] alkylamines-(AdBOAA) and the 3-cyclopentyl-adamantyl-aminesor alkylamines or alkyiphenyl amines (AdCP), halogenated, ormethoxylated on their substitutes and their pharmaceutically acceptablesalts of the racemics, enantiomers and diastereoisomers and also thecharacterized by the inventor as a ligands generics Quinacrine (QNC),Methylene blue (MB), Astemizole (AST) and their related analogues,withprototypical anticancer and antimetastatic properties associated withanalgesic activity against the neuropathic pain, generally, and alsothose induced by the clinically used anticancer drugs. At lower dosesthe above compounds and drugs exhibited prototypical cytoprotectiveproperties associated with cytoregenerative activity developed byantagonism of the endoplasmic reticulum stress (ER stress) viaactivation of the sigma receptors in the connecting membrane between ERand mitochondrion.

The present invention concretize the concept of bio-modulatory activitywith the original σ-receptor ligands AL, AE, AdBAA, AdBOAA, AdCP andthe, recently characterized by the inventor as σ ligands, generics:QNC,MB,AST and also their related analogues with a new and sophisticatedmechanism including the disclosed orthosteric and allosteric modulationsbut, principally, the activation of their σ-1 receptors (chaperones) inthe dysregulated cells of the degenerative diseases characterized byhyper activation of the apoptotic process or, in cancer cells, thedeactivation of these chaperones, at high doses of the above ligands,where they abolish the defensive inhibition of the apoptotic processusin the cancer cells.

In vitro and in vivo experiments clearly demonstrated the principalinteraction of AL, AE, AdBAA, AdBOAA, AdCP and also QNC,MB and AST overthe σ-1, σ-2 receptors from sub-nanomolar up to micromolar levels ofaffinity along with their regulatory roles over the G-protein coupledreceptors (GPCRs) and sodium (Na), calcium (Ca), potassium (K) andchloride (Cl) ion channels and, principally, on the pathways of the ERand mitochondrion which determinate the protection of the cells in thedegenerative diseases and the apoptosis of the cancer cells.

Thus, in contrast to the precedent patents of the inventor, the aboveinvention displaced the area of the putative application of thesemolecules from the symptomatic to the therapeutic use because, speciallyfor the cytoprotecting and cytoregenerating properties of the followingclaims the activity of the molecules concern the intracellular targetsand pathways of pathogenesis and not the activity of superficial cellmembrane receptors, as it is usually the case with the symptomaticdrugs: neuroleptics, anti depressive drugs and cholinergics for,respectively, psychosis, depression and Alzheimer.

On the other hand, recent experiments in vivo demonstated theprototypical profile of the above σ ligands which associated theanticancer activity with the antagonism of pain or neu-ropathic pain andalso those originated from the anticancer treatments with the clinicallyused anticancer drugs and specially with taxanes, platinum agents andvicristine. Thus, the inven-tion of this prototypical activity,supported the synergistical use of the above σ ligands with theclinically active anticancer drugs—as it was recently demonstrated withxenograft expe-riments on SCID mice and also, with neuropathic painprotocols, on CD-1 and Balb-C mice—with simultaneous antagonism, by theabove Cr ligands, of the painful effects and disability in-duced by theclinically used anticancer drugs.

Finally, their ability to regulate cellular apoptotic processes, throughER-mitochondrial σ-1 and σ-2 receptors, associated with thestereochemical re-articulation of the membrane sphingolipides, inducedby the above ligands, supported the antiviral activity of the abovecompounds (more specially those containing adamantyl(s)),which wasdemonstrated experimentally in vitro.

EXAMPLES

A) Sigma (σ)-1 Agonists with Bio-Modulatory Properties and withAntagonistic Action on the Apoptotic Processes of the Cells.

-   -   AE14: TETRAHYDRO-N,N,-DIMETHYL-5,5-DIPHENYL-3-FURANOMETHANAMINE

AE14 is a selective ligand for σ-1 receptors with a nanomolar affinity(pKi=7,6) while the affinity for σ-2 receptors is more than 100 timeslower [over 10 micro-Moles (10 μM): pKi<5]. The above molecular profileis the basis for AE14 modulatory role over the G-protein coupledreceptors (GPCRs) and its pharmacological properties: anti-amnesicaction (anta-gonistic action against scopolamine and dizocilpine(MK-801) induced amnesia or amyloid peptide β25-35 (Aβ25-35), at lowdoses (from 0.03 mg/kg administered per os (po), in mice). This uniquenew profile of AE14: anti-apoptotic via agonistic sigma-1 regulation ofthe volume regulated chloride channels (VRCC), anti-oxidative stress viaagonistic action on the sigma-1 receptors of the ER-mitochondrion.

Recent experiments amplified the above disclosed results anddemonstrated the putative cytoprotective and cytoregenerative activityof AE14 and the relative analogues, by triggering the activity of the σchaperones in the ER-mitochondrion,in protocols of acute or chroniccellular stress (in vitro and in vivo on mice) using oligopeptides orstreptozocin which initiate ER stress and cytotoxic activity byapoptosis. In the above protocols, AE14 antagonized the ER stressextending its application area to the intimate causes: dysregulation ofthe σ chaperones regulated alarm pathways to mitochondrion,and thereforeto all the cytodegenerative diseases.

-   -   TETRAHYDRO-N,N-DTIVIETHYL-2,2-DINIENYL-3-FURANOMETHANAMINE

Similar pharmacological profile as to AE14: smaller affinity for σ-1receptors (pKi=6,3) and with no affinity for σ-2 receptors.

Therefore, AE37, as a selective σ-1 agonist with anti-apoptotic action,was recently studied in vitro and in vivo on mice in protocols ofchronic ER and cellular stress and exhibited high anti-apoptoticproperties and putative cytoprotective and cytoregenerative activity.

-   -   AE37Met:TETRAHYDRO-N-METHYL-2,2-DIPHENYL-3-FURANOMETHANAMINE

AE37 Met is the only metabolite of AE37 and is slightly more active thanAE37 for the properties mentioned above but it mainly concerns thecytoprotective action, especially against acute intracellular(ER-mitochondrion) stress and then against the ischemic syndromes(brain, heart), in relevance with its exceptional protection againstER-mitochondrion anoxia and oxidative stress caused bypentylenetetrazole (PTZ), in vitro or in vivo, on mice, following itsprotection against the PTZ-induced tonic crises.

B) Weak σ-1 Agonists or σ-1 Antagonists with Agonistic σ-2 Component andAgonistic Action to the Apoptotic Processes of the Cells.

-   -   The following molecules are representatives of the chemical        families that concern the present invention:    -   1) MAL: DIHYDRO-4-(D1METHYLAMINOMETHYL) SPIRO        {FURANE-2(5H),2′-TRICYCLO[3.3.1.1_(3,7)] DECAN}-5-ONE 6—    -   2) AdAE: (DIHYDRO-4-(DIMETHYLAMINOMETHYL)SPIRO        {FURANE-2(3H),2′-TRICYCLO[3.3.1.1_(3,7)] DECANE}    -   3) AdPhAL: 5-(TRICYCLO[3.3.1.1_(3,7)]        DEC-1-YL)-DIHYDRO-3-[DIMETHYLAMINOMETHYL)-5-PHENYLFLTRAN-2(3H)-ONE—    -   4) AdPhAE: 5-(TRICYCLO[3.3.1.1_(3,7)]        DEC-1-YL)-TETRAHYDRO-N,N-DIMETHYL-5-PHENYL-3-FURANEMETHANAMINE—    -   5)        AL142Me:(α-(DINIETHYLAMENOETHYL)-γ,γ-DIPHENYL-γ-BUTYROLACTONE)    -   6) AdBPA:γ-(1-ADAMANTYL)PRENYL-N,N-DIMETHYLPROPANAMINE    -   7) AdBPP: γ-(1-ADAMANTYL)PHENYL-PROPYLPIPERIDINE    -   8) AdBOPP: [α-(ADAMANTYL-1)BENZYLOXY-2] PROPYLPIPERIDINE    -   9) AdBOEA (Me): N,N        DIMETHYL-[α-(ADAMANTYL-1)BENZYDRYLOXY-2-ETHYLAMINE    -   10) -Ad(Me)CP:        3-CYCLOPENTYL-N-METYL-1-TRICYCLO[3.3.1.1_(3,7)]DECANEMETHANAMINE    -   11) Quinacrine (QNC)    -   12) Methylene blue (MB)    -   13) Astemizole (AST)    -   (see also, in appendix, undescribed derivatives of the above        chemical families)

All molecules present strong chemical affinities (sub-nanomolar tomicromolar) for σ-1 receptors with antagonistic action (except AdAE andAdPhAE which are weak agonists) and with nanomolar to micromolaraffinities for σ-2 receptors and agonistic action. Therefore, allmolecules above and their derivatives are pro-apoptotic and only at verylow concentrations or doses are anti-apoptotic. All molecules above aretoxic to the cancer cells : colorectal, prostate, ovarian renal,pancreas, lung, gliomas, glioblastomas leukemia, lymphomas, melanomas,sarcomas and hepatoma, with antiproliferative and cytotoxicconcentrations usually lower than 10 microMolar (μM),for more than 50 ofthe above derivatives. Outstanding cytotoxicity, in vitro and thecorresponding in vivo anticancer activity in mice xenografts, is for thefirst time disclosed for QNC and AST : 3.0-5.0 μM for all the abovecancer cells and an outstanding in vivo activity of QNC on primary (drugresistant) melanoma, in which Dacarbazine is inactive, or MB : lowerthan 1.0 μM in vitro for primary (drug resistant) melanoma or leukaemiaand lung and close to 2.0 μM in breast, colon and glioblastomasconfirmed in vivo, specially in primary melanoma. All these moleculesmake excellent and prototypical anticancer drug candidates due to theiraptitude to, simultaneously, antagonize the neurogenic and, moreexceptional, the neuropathic and inflammatory pain as recentlydemonstrated by the appropriate experimental protocols: hot plate,formalin, von Frey fibers and, principally, the protocols based onprevious administration of paclitaxel, oxaliplatine and streptozocinwhich concern more specifically the neuropathic pain originating fromchronic administration of anticancer drugs, from diabetes or otherneurotoxic stimulus. The pharmacological profile of the above moleculesis totally prototypical and original giving the possibility of atherapeutic synergy with the clinically used anticancer drugs withsimultaneous antagonism and protection against the most grave secondaryeffects i.e., neuropathic pain and disability induced by the clinicallyused anticancer drugs.

-   -   1) Pouli N., Fytas G., Foscolos G., Kolocouris N., Marakos P.,        and Vamvakides A. Synthese et etude pharmacologique des        adaman-tylbenzenepropanamines et propenamines. Annales        Pharmaceutiques Francaises, 1995, 53, No 4, pp 163-169    -   2) Fytas G., Marakos P., Kolocouris N., Foscolos G., Pouli N.,        Vamvakides A., Ikeda S., De Clercq E.        3-cyclopentyl-1-aclamantamines and adamantanemethanamines.        Antiviral activity evaluation and convulsion studies. Farmaco        1994, 49, No 10, pp 641-647    -   3) Vamvakides Alexandre:        Tetrahydro-N,N-dimethyl-2,2-diphenyl-3-furanemethanamine, its        enantiomers and their pharmaceutically acceptable acid addition        salts. WO97/30983 and patent 1002616 (GR)

1-8. (canceled)
 9. A method of using sigma(σ) receptor ligands, saidmethod comprising the steps of: providing a compound comprising asigma(σ)-receptor ligands and a sigma(σ) ligands generics, said sigma(σ)ligands generics being selected from the group consisting of Quinacrine,analogues of Quinacrine, Methylene blue, analogues of Methylene blue,Astemizole, and analogues of Astemizole; and using said compound forpreparation of pharmaceuticals with anticancer, antimetastatic andantiviral activity associated with analgesic properties.
 10. The methodaccording to claim 9, wherein said sigma(σ)-receptor ligands beingselected from the group consisting of (mono- ordi-alkylaminoalkyl)-γ-butyrolactones), aminotetrahydrofuranes,enantiomers or diastereoisomers of aminotetrahydrofuranes, (1-adamantyl)benzene alkylamines, enantiomers or diastereoisomers of (1-adamantyl)benzene alkylamines, N,N di-alkyl α-[(adamantyl-1) benzyloxy-2]alkylamines, enantiomers or diastereoisomers of N,N di-alkylα-[(adamantyl-1) benzyloxy-2] alkylamines, 3-cyclopentyladamantyl-amines or -alkylamines or -alkyl phenylamines, enantiomers ordiastereoisomers of 3-cyclopentyl adamantyl-amines or -alkylamines or-alkyl phenylamines, and their pharmaceutically acceptable salts. 11.The method according to claim 10, wherein said compound furthercomprising at least one pharmaceutically acceptable excipient.
 12. Amethod of using sigma(σ) receptor ligands, said method comprising thesteps of: providing a compound comprising a sigma(σ)-receptor ligandsand a sigma(σ) ligands generics, said sigma(σ) ligands generics beingselected from the group consisting of Quinacrine, analogues ofQuinacrine, Methylene blue, analogues of Methylene blue, Astemizole, andanalogues of Astemizole; and using said compound for preparation ofpharmaceuticals with analgesic activity against neuropathic pain. 13.The method according to claim 12, wherein said sigma(σ)-receptor ligandsbeing selected from the group consisting of (mono- ordi-alkylaminoalkyl)-γ-butyrolactones), aminotetrahydrofuranes,enantiomers or diastereoisomers of aminotetrahydrofuranes, (1-adamantyl)benzene alkylamines, enantiomers or diastereoisomers of (1-adamantyl)benzene alkylamines, N,N di-alkyl α-[(adamantyl-1) benzyloxy-2]alkylamines, enantiomers or diastereoisomers of N,N di-alkylα-[(adamantyl-1) benzyloxy-2] alkylamines, 3-cyclopentyladamantyl-amines or -alkylamines or -alkyl phenylamines, enantiomers ordiastereoisomers of 3-cyclopentyl adamantyl-amines or -alkylamines or-alkyl phenylamines, and their pharmaceutically acceptable salts. 14.The method according to claim 13, wherein said compound furthercomprising at least one pharmaceutically acceptable excipient.
 15. Amethod of using sigma(σ) receptor ligands, said method comprising thesteps of: providing a compound comprising a sigma(σ)-receptor ligandsand a sigma(σ) ligands generics, said sigma(σ) ligands generics beingselected from the group consisting of Quinacrine, analogues ofQuinacrine, Methylene blue, analogues of Methylene blue, Astemizole, andanalogues of Astemizole; and using said compound for preparation ofpharmaceuticals selected from the group consisting of pharmaceuticalsacting synergistically with clinically used anticancer drugs andantagonizing neuropathic pain induced by the anticancer drugs,pharmaceuticals with cytoprotective activity against pathogenesis ofcytodegenerative diseases, pharmaceuticals with cytoprotective andcytoregenerative activity, and pharmaceuticals with protective activityagainst the pathogenesis of inflammatory and neuropathic pain.
 16. Themethod according to claim 15, wherein said sigma(σ)-receptor ligandsbeing selected from the group consisting of (mono- ordi-alkylaminoalkyl)-γ-butyrolactones), aminotetrahydrofuranes,enantiomers or diastereoisomers of aminotetrahydrofuranes, (1-adamantyl)benzene alkylamines, enantiomers or diastereoisomers of (1-adamantyl)benzene alkylamines, N,N di-alkyl α-[(adamantyl-1) benzyloxy-2]alkylamines, enantiomers or diastereoisomers of N,N di-alkylα-[(adamantyl-1) benzyloxy-2] alkylamines, 3-cyclopentyladamantyl-amines or -alkylamines or -alkyl phenylamines, enantiomers ordiastereoisomers of 3-cyclopentyl adamantyl-amines or -alkylamines or-alkyl phenylamines, and their pharmaceutically acceptable salts. 17.The method according to claim 16, wherein said compound furthercomprising at least one pharmaceutically acceptable excipient.